Department

First Thesis Advisor

Keywords

Abstract

This body of work details the exploitation of an incredibly powerful neural culture system, which enables the in vitrostudy of events involved in peripheral nervous system (PNS) development. Using a myelinating dorsal root ganglion (DRG) explant culture system, node of Ranvier formation and maintenance and the associated generation and maturation of myelin segments was examined. In addition, Schwann cell (SC) development, dynamics, and migration were extensively studied.

First, in characterizing these cultures, the discrete axonal localization of specific ankyrin isoforms was revealed. Ankyrins are peripheral membrane proteins that immobilize classes of integral membrane proteins to the spectrin based-membrane skeleton. Ankyrins interact with proteins such as the voltage-dependent/gated sodium channel (vgsc) and members of the L1 family of cell adhesion molecules. These interactions are physiologically relevant to the formation of membrane specializations involved in axon guidance and the initiation and propagation of action potentials.

We examined ankyrinB and ankyrinG expression in cultured DRG explants, which allowed visualization of individual axons. AnkyrinB and ankyrinG exhibited differential localizations to specific axonal populations. This was evident as early as one day in vitro and persisted over time. In mature pre-myelinated cultures, axons having an apparent diameter of less than 1 µm predominantly expressed ankyrinB, whereas axons having a diameter greater than or equal to 1 µm predominantly expressed ankyrinG (based on immunocytochemical reactivity). When myelination was induced, ankyrinGwas appropriately localized to sites of nodal development flanked by myelinating glial processes in the large caliber axons. These observations suggest that axons destined for myelination may express a distinct complement of peripheral, and perhaps integral, membrane proteins as compared to those observed in non-myelinated axons. These distinguishing features may play a role in the selection of axons for myelination.

This work was followed with defining the role axo-glial interactions play in organizing domains along the axon being myelinated. Nodes of Ranvier are specialized, highly polarized axonal domains crucial to the propagation of saltatory action potentials. In the PNS, axon-glial cell contacts have been implicated in SC differentiation and the formation of nodes of Ranvier. SC microvilli establish axonal contact at mature nodes, and their components have been observed to localize early to sites of developing nodes. However, a role for these contacts in node formation remains controversial.

Using the myelinating explant culture system, we observed that SCs reorganize and polarize microvillar components, such as the ezrin-binding phosphoprotein 50kDa (EBP50)/regulatory co-factor of the sodium-hydrogen exchanger isoform 3 (NHERF-1), actin, and the activated ezrin, radixin, and moesin (ERM) family of proteins, concomitant with myelination in response to inductive signals. These components were targeted to the SC distal tips where live cell imaging revealed novel, dynamic growth cone-like behavior. Further, localized activation of the Rho signaling pathway at SC tips gave rise to these microvillar component-enriched “caps” and influenced the efficiency of node formation.

Extending these findings, a more profound examination of SC dynamics was undertaken. This was a particularly important experimental transition, as SC motility is crucial in the development and regeneration of the PNS. The seemingly equivalent bipolarity of mature SCs represents a conundrum in terms of directed motility. Fluorescence time-lapse microscopy of transfected SCs within the myelinating DRG explants revealed a novel cycling of these cells between static, bipolar and motile, unipolar morphologies via asymmetric process retraction and extension. Concentrations of PIP2 (phosphatidylinositol (4,5)-bisphosphate), activated ERMs, and EBP50 delineated the transitory asymmetry associated with the generation and neuron-like migration of the unipolar cell. EBP50 over-expression enhanced unipolar SC migration, suggesting a new role for this adaptor protein in cell motility. Further, the ERMs themselves were found to be essential to both motility and process dynamics with ERM disruption yielding a dysfunctional, multipolar SC phenotype. We propose this novel form of motility may be associated with the correct alignment and spacing of SCs along axons prior to elaboration of the myelin sheath.

These compiled studies present significant advances in understanding and examining axo-glial interactions in the PNS. This work establishes the foundation for further, novel exploration of normal PNS development and the regeneration and repair mechanisms involved in PNS injury and disease states.

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